The present invention relates to methods for detection of anti-human leukocyte antigen (HLA) reactive antibodies. Individuals may be sensitized to HLA antigens during pregnancy, or by blood transfusion or previous organ grafts. Testing to determine sensitivity to HLA alleles is relevant to tissue and organ transplantation where the presence in the recipient of antibodies against HLA antigens of the donor (donor specific crossmatch) is predictive of a high risk of graft rejection. It is a standard practice in the transplant field to test all potential recipients against a panel of HLA antigens selected to represent a human population and the percentage of HLA alleles against which the serum is reactive is determined. In this panel reactive antibody (PRA) testing reaction of a patient""s serum against a high percentage of HLA alleles present in a normal human population is predictive of a high risk of graft rejection.
Methods known in the art for HLA testing include the complement-dependent lymphocytotoxicity (CDC) test in which serum from a recipient is incubated with donor or panel lymphocytes followed by incubation with complement. The level of cytotoxicity is then estimated by discriminating between dead and viable cells using a dye. This method is labor intensive, requires viable cells, may be nonspecific and requires a subjective evaluation.
Pouletty et al. U.S. Pat. No. 5,223,397 discloses methods for testing HLA compatibility between a donor and a recipient comprising the steps of adding blood from the donor to a substrate having anti-HLA antibodies bound thereto and incubating for sufficient time for soluble HLA antigens present in the blood to bind to the antibodies or ligand. Blood from the recipient is then added to the solid substrate whereby any antibody specific for any HLA antigens bound to the solid substrate may become bound. The detection of an absence of antibodies from the recipient""s blood to the HLA antigen is indicative of a cross-match.
Zaer et al., Transplantion 63: 48-51 (1997) discloses use of an ELISA using HLA class I molecules purified from pooled platelets to detect anti-HLA antibodies. The reference reports that in patients found to be unsensitized, the incidence of false-positive results was less for ELISA testing than for panel studies. In patients who were highly sensitized, both tests performed equally well, whereas discordant results were registered mainly in cases of mild sensitization. In such cases, the incidence of false-negative results was higher for ELISA testing than for panel studies.
Of interest to the present invention are assay methods making use of flow cytometry. Wilson et al., J. Immunol. Methods 107: 231-237 (1988) disclose the use of polyacrylamide microspheres coupled with cell membrane proteins in immunofluorescence assays for antibodies to membrane-associated antigens. The method is said to make possible the rapid flow cytometric analysis of plasma membrane antigens from cell populations that would otherwise be unsuitable for use in flow cytometry. Scillian et al., Blood 73: 2041-2048 (1989) disclose the use of immunoreactive beads in flow cytometric assays for detection of antibodies to HIV. Frengen et al., Clin. Chem. 40/3: 420-425 (1994) disclose the use of flow cytometry for particle-based immunoassays of ce-fetoprotein (AFP). This reference further reports the ability of serum factors to cross-link labeled mouse monoclonal antibodies of irrelevant specificity to different particle types coated with various immunoglobulins.
Flow cytometry methods using lymphocytes are also known but suffer with difficulties because of the activity of auto-antibodies. See Shroyer et al., Transplantation 59:626-630 Moreover, when using flow cytometry with lymphocytes, use of ten or more different lymphocytes tends to result in confusing signals. As a consequence, studies using lymphocytes have been limited by presenting a small panel of HLA antigens that do not effectively simulate the distribution of HLA antigens in a normal human population.
Sumitran-Karuppan et al., Transplantation 61: 1539-1545 (1996) discloses the use of magnetic beads which use an anti-HLA capture antibody to immobilize a variety of soluble HLA antigens pooled from 80 to 100 individuals on each bead. The beads can then be directly added to patient serum for efficient absorption of HLA antibodies. The reference discloses visualization of antibody binding to the antigen-coated beads using flow cytometry. The reference suggests that this development will allow testing for antibody specificity for crossmatching purposes and for the screening of panel-reactive antibodies. The methods of Sumitran-Karuppan are limited, however, because the pooling of antigens causes sensitivity to certain rare HLA antigens. Moreover, the method is not capable of detecting the percentage of PRA.
Accordingly, there remains a need in the art for improved methods of HLA typing including methods for determination of percentage of PRA which is rapid, convenient and accurate.
The present invention generally relates to methods for detection of panel reactive antibodies in serum of a subject against HLA antigens including Class I and class II antigens. Specifically, the method comprises the steps of providing a collection of microbeads of different subtypes wherein microbeads of at least one subtype each present HLA antigens derived from a cell population presenting the same HLA antigens which is preferably but not necessarily a single lymphocyte cell line; adding serum from a subject to said collection of microbeads; incubating said serum and microbeads for sufficient time for anti-HLA antibodies in said serum to bind to said HLA antigens; removing said serum components which do not specifically bind with said HLA antigens presented on said microbeads; incubating said microbeads with a labeled ligand capable of binding with anti-HLA antibodies bound to said HLA antigens; removing said labeled ligand which is not bound to said anti-HLA antibodies; and detecting the presence of labeled ligand bound to said anti-HLA antibodies antigens by flow cytometry. According to a preferred aspect of the invention, microbeads of each subtype present HLA antigens derived from a cell population presenting the same HLA antigens which can be derived from a single human individual and may be lymphocytes, platelets or another cell population which present HLA antigens. A preferred source is a single lymphocyte cell line. According to preferred methods of the invention, the panel of HLA antigens is selected to simulate distribution of Class I and/or Class II HLA antigens in a normal human population and also allows most rare antigens to be represented. According to particularly preferred methods, the panel comprises 54 different Class I HLA antigens obtained from 30 different cell lines. Alternatively, the panel can preferably comprise 22 different Class II HLA antigens preferably selected from 15 to 30 different cell lines. While the use of greater numbers of cell lines as sources for antigens can more closely simulate the natural distribution of antigens there is also a desire to minimize the number of cell lines used to promote greater sensitivity of the assay. Nevertheless, it will be within skill in the art to balance these factors in specifically designing an assay format.
The microbeads of the invention may be made of a wide variety of suitable materials with latex beads such as those available from Spherotech Inc. being particularly preferred. The microbeads may be of any size suitable for analysis by flow cytometry with diameters ranging from about 2 xcexcm to about 15 xcexcm being preferred and particles with diameters of about 3 xcexcm being particularly preferred for beads presenting Class I HLA antigens and diameters of about 5 xcexcm being preferred form beads presenting Class II HLA antigens. The invention further provides methods wherein microbeads of at least one HLA subtype differ from microbeads of at least one other HLA subtype by being selected to have different diameters or by having different labels such as are known to the art with fluorescent labels being preferred.